1. Field of the Invention
The present invention relates to a technique for monitoring and recording the activity of organism tissue in vivo, that is, keeping an organism specimen alive and monitoring a physiological change, in particular, it relates to a technique for enabling researchers to efficiently detect a specific portion to be the subject of interest in an organism specimen.
2. Description of the Prior Art
As a method for recording the activity of organism specimen, which receives a stimulus from the out side, that is, as a method for monitoring the change in physiological functions of organism tissue in vivo, an electrophysiological technique is conventionally used to extracellularly measure the action potential of nerve cells in the vicinity of a probe such as a tungsten electrode, the tip of which is inserted into a specific portion of organism specimen.
However, in case of recording the activity of organism tissue in vivo, since the portion wherein an observation subject such as the nerve cell exists can be roughly detected, a troublesome operation, which needs preparation of tissue slices as histochemical specimens to ascertain whether or not the monitoring area inserted with the probe is correct, is required after recording the activity and collecting the resultant data. For example, using a mouse as a specimen for recording the action potential of nerve cells at a specific portion of the brain in vivo, the following sequential operation is executed, roughly detecting a target portion according to the map information of mouse brain empirically obtained, inserting an electrode into the portion to record the action potential of nerve cells, taking out the brain to prepare section specimens for histochemistry, and estimating the electrophysiological data obtained and the portion of the brain defined.
As mentioned above, in case of conventional recordings of the activity at a specific portion of organism specimen in vivo, since the portion can be roughly detected, the section specimens of organism tissue are required to confirm the recorded portion to be correct after collecting electrophysiological data of interest. Therefore, if the recorded portion is not a desirable one, the above-mentioned operation should be repeated until the acquired result is a satisfactory one.
Accordingly, an object of the present invention is to provide an apparatus for detecting a specific portion and recording the activity of cells in an organism specimen in vivo, a method capable of finding the specific portion easily and a method capable of recording physiological activities in the specific portion accurately.
In order to solve problems mentioned above, the first aspect of the present invention is a method for detecting the specific portion, which comprised of the following steps; expressing a fluorescent substance (such as GFP: green fluorescent protein) at the specific portion of organism specimen, inserting an optical fiber into the organism specimen in vivo, performing irradiation with exciting rays through an optical fiber, and monitoring to detect the specific portion emitting the resultant fluorescence through the optical fiber.
According to the method for detecting the specific portion of organism specimen, an insertion of optical fiber into the specimen makes it easy to find a target portion by examining whether or not the fluorescence can be seen through the optical fiber.
The second aspect of the present invention is a method for monitoring the fluorescence from the specific portion with the fluorescent substance excited by the exciting rays through the optical fiber using a fluorescence microscope while irradiating the exciting rays from the fluorescence microscope to the inside of the organism specimen through the optical fiber.
According to the method for detecting the specific portion, since a spell of operation irradiating the inside of organism specimen with exciting rays and monitoring the fluorescence from the special portion excited by exciting rays through the optical fiber can be efficiently executed by use of a fluorescence microscope, the specific portion of organism specimen can be unearthed easier compared with the case of the first aspect.
Furthermore, the third aspect of the present invention is a method of physiological measurement comprised of the following steps; expressing a fluorescent substance at the specific portion of organism specimen, inserting an optical fiber into the specimen, performing irradiation of exciting rays to the inside of organism specimen through the optical fiber, monitoring the fluorescence from the specific portion excited by the exciting rays through the optical fiber, inserting a probe into the specific portion or in the vicinity thereof, and recording physiological activities at the specific portion or in the vicinity thereof via the probe.
According to the method of physiological measurement, since the specific portion of organism specimen can be easily unearthed only by inserting the optical fiber and examining whether or not the fluorescence can be seen through the optical fiber, the activity of organism specimen in vivo can be easily recorded by inserting the probe into the specific portion or in the vicinity thereof.
Moreover, the fourth aspect of the present invention is a method of physiological measurement according to the third aspect, wherein the specific portion is unearthed by monitoring the fluorescence from the specific portion excited by the exciting rays through the optical fiber using a fluorescence microscope while by irradiating exciting rays to the inside of organism specimen through the optical fiber using the fluorescence microscope, and the activity of the specific portion or in the vicinity thereof is measured by inserting the probe into the specific portion unearthed or in the vicinity thereof.
According to the method of physiological measurement, since both irradiation of exciting rays to the inside of organism specimen and monitoring of fluorescence excited by the exciting rays through the optical fiber can be efficiently executed by use of a fluorescence microscope, so that the specific portion of organism specimen can be further easily found out, an experiment for recording the activity of organism specimen in vivo can be executed easier compared with the case of the third aspect of inserting the probe into the specific portion or in the vicinity thereof for measurement.
Furthermore, the fifth aspect of the present invention is an apparatus comprised of an optical fiber which is inserted into an organism specimen, that is, the first optical system for irradiation of exciting rays to the inside of organism specimen through the optical fiber and the second optical system for monitoring of fluorescence excited by the exciting rays through the optical fiber.
According to the specific portion detecting apparatus, by inserting the optical fiber into the organism specimen with an fluorescent substance expressed at the specific portion for recording the activity, irradiating the exciting rays to the inside of the organism specimen through the optical fiber by the first optical system, and observing the fluorescence excited by the exciting rays through the optical fiber by the second optical system, the specific portion of the organism specimen can be easily found out. That is, by using the specific portion detecting apparatus according to the fifth aspect, a specific portion of an organism specimen can be easily found out by the specific portion detecting method according to the first aspect. Therefore, by inserting a probe into the found out specific portion or in the vicinity thereof for measurement, the activity recording experiment of the organism specimen in vivo can be easily executed.
Moreover, a sixth aspect of the present invention is a specific portion detecting apparatus comprising a fluorescence microscope for irradiating the exciting rays to an organism specimen through an objective lens and catching the fluorescence excited by the exciting rays by the objective, an optical fiber with one end to be inserted into the organism specimen, and an optical fiber holding device for holding the other end surface of the optical fiber, facing to the objective.
According to the specific portion detecting apparatus, since the operation of irradiating the exciting rays to the inside of the organism specimen through the optical fiber and observing the fluorescence excited by the exciting rays through the optical fiber can be efficiently executed using the fluorescence microscope, the specific portion of the organism specimen can be easily found out. That is, using the specific portion detecting apparatus according to the sixth aspect, a specific portion of an organism specimen can be easily found out by the specific portion detecting method according to the second aspect. Therefore, by inserting a probe into the specific portion found out or in the vicinity thereof for measurement, the activity recording experiment of the organism specimen in vivo can be easily executed.
Furthermore, the seventh aspect of the present invention is the specific portion detecting apparatus according to the sixth aspect, wherein the optical fiber holding device is fixed detachably on a stage of the fluorescence microscope. The stage herein denotes a placing base provided below the objective of the fluorescence microscope for placing a sample, movably in the horizontal direction and further in the vertical direction.
The specific portion detecting apparatus can be used as the specific portion detecting apparatus according to the sixth aspect with the optical fiber holding device fixed to the stage of the fluorescence microscope so as to hold the optical fiber, and it can be used as an ordinary fluorescence microscope with the optical fiber holding device detached from the stage of the fluorescence microscope.
Moreover, the eighth aspect of the present invention is the specific portion detecting apparatus according to the seventh aspect, wherein the optical fiber holding device comprises a flat plate-like pedestal fixed detachably to the stage of the fluorescence microscope, and a gripping device fixed to the pedestal for holding the end portion of the optical fiber perpendicular to the stage.
The specific portion detecting apparatus can be used as the specific portion detecting apparatus according to the sixth aspect with the pedestal of the optical fiber holding device fixed to the stage of fluorescence microscope so as to hold the optical fiber by the gripping device, and it can be used as an ordinary fluorescence microscope with the optical fiber holding device detached from the stage of fluorescence microscope.
Furthermore, the ninth aspect of the present invention is an optical fiber holding device comprising a flat plate-like pedestal fixed detachably to the stage of the fluorescence microscope, and a gripping device fixed to the pedestal for holding the end portion of the optical fiber perpendicular to the stage.
According to the optical fiber holding device, since it can be used as the specific portion detecting apparatus according to the sixth aspect with the pedestal fixed to the fluorescence microscope and the optical fiber held by the gripping device, a specific portion of an organism specimen can be easily found out using the fluorescence microscope.
As the pedestal in the eighth and ninth aspects, the plate having a size and shape fixed by a sample applanator provided on the stage, such as a glass plate used as a slide glass or a preparation, or a metal plate or a synthetic resin plate having the same size as the preparation can be preferably used.